Blood Groups | Zoology Optional Notes for UPSC PDF Download

ABO Blood Groups

1. Antigens:

   • ABO blood group system has two antigens on the surface of red blood cells (RBCs): A and B.
   • The presence or absence of these antigens classifies blood into four types: A, B, AB, and O.

2. Antibodies:

   • In addition to the antigens, there are two antibodies in the plasma:
     • Anti-A antibodies
     • Anti-B antibodies
   • Individuals with blood type A have anti-B antibodies, and those with blood type B have anti-A antibodies.
   • Blood type AB has both A and B antigens and lacks anti-A and anti-B antibodies.
   • Blood type O has no A or B antigens but has both anti-A and anti-B antibodies.

Development at Birth

• ABH antigens develop early in fetal life (around day 37) but do not significantly increase during gestation.
• Newborn red cells carry 25-50% of the antigenic sites found on adult RBCs.
• A or B antigen expression fully develops between 2-4 years of age and remains constant throughout life.

Expression of ABO Antigens:

• While considered RBC antigens, ABO blood group antigens are expressed on various human tissues and most epithelial and endothelial cells.
• ABH antigens are found in humans and some apes (chimpanzees, gorillas) on both red blood cells and in secretions.
• In other mammalian species, these antigens are found only in secretions.

Anti-A and Anti-B Antibodies

• Anti-A and Anti-B antibodies naturally appear in the plasma at around 3 to 6 months of age, even without exposure to foreign blood group antigens.
• This natural production of antibodies is known as isoagglutinin production.

Clinical Significance:

• ABO blood groups are crucial for blood transfusions to avoid transfusion reactions.
• Incompatible blood transfusions can lead to agglutination (clumping) of red cells, causing serious complications.

Other Blood Group Systems:

• Besides ABO and Rh, there are minor blood group systems such as Kell, Kidd, Duffy, etc.
• These systems involve different antigens and antibodies and are important for specific transfusion scenarios.

Inheritance of ABO Blood Groups

• Mendelian Principles:

  • ABO blood group inheritance follows Mendelian principles.
  • Blood group antigens are "codominant," meaning if the gene is inherited, it will be expressed.

• Allelic Genes:

  • There are three allelic genes involved: A, B, and O.
  • These genes determine the ABO blood group.

• Genotypes and Phenotypes:

  • Two genes are inherited, one from each parent.
  • Individuals who are blood type A or B may be homozygous or heterozygous for the antigen.
    • Heterozygous: AO or BO
    • Homozygous: AA or BB
  • Phenotype is the actual expression of the genotype (e.g., blood type A).
  • Genotype refers to the actual inherited genes, determined by family studies (e.g., AO).

ABO and H Antigen Genetics

• Loci and Antigen Control:

  • Genes at three separate loci control the occurrence and location of ABO antigens.
  • The presence or absence of ABH antigens on the red cell membrane is controlled by the H gene.
  • The presence or absence of ABH antigens in secretions is indirectly controlled by the Se gene.
    • H gene – H and h alleles (h is an amorph)
    • Se gene – Se and se alleles (se is an amorph)
    • ABO genes – A, B, and O alleles

• Homozygous and Heterozygous Combinations:

  • Individuals can have different combinations of these alleles, leading to various blood types.
  • For example, someone with the genotype AO has blood type A, and someone with the genotype BB has blood type B.

• Aberrant Genotypes:

  • While some aberrant genotypes do occur, they are very rare.
  • Understanding the basic principles of inheritance is crucial for predicting blood group outcomes based on parental genotypes.

• Importance of ABO Blood Group Knowledge:

  • Knowledge of ABO blood group genetics is essential for blood transfusions and organ transplants to ensure compatibility and prevent adverse reactions.

H Antigen and ABO Antigens Formation

• H Antigen:

  • The H gene codes for an enzyme called fucosyltransferase.
  • This enzyme adds the sugar fucose to the terminal sugar of a precursor substance, which is formed on an oligosaccharide chain.

• Foundation for A and B Antigens:

  • The H antigen serves as the foundation upon which A and B antigens are built.
  • A and B genes code for enzymes that add immunodominant sugars to the H antigen.
  • Immunodominant sugars are present at the terminal ends of the chains and determine the ABO antigen specificity.

• Formation of H Antigen:

  • The precursor substance (proteins and lipids) is formed on an oligosaccharide chain, with the basic structure remaining the same.
  • The H antigen is formed by the addition of fucose to this precursor substance.
  • The H antigen acts as a precursor for the further addition of sugars to create A and B antigens.

Structure:

• RBC Precursor Substance:
  Glucose - Galactose - N-acetylglucosamine - Galactose
• Formation of H Antigen:
  RBC - Fucose - Glucose - Galactose - N-acetylglucosamine - Galactose

A and B Antigens:

• The "A" gene codes for an enzyme (N-acetylgalactosaminyltransferase) that adds N-acetylgalactosamine to the terminal sugar of the H antigen.
• The "B" gene codes for an enzyme that adds D-galactose to the terminal sugar of the H antigen (D-galactosyltransferase).

Characteristics of Bombay Phenotype

• Discovery:

  • First reported by Bhende et al in Bombay in 1952.

• Frequency:

  • Estimated frequency is about 1 in 7600 in Bombay.

• Antigen Absence:

  • Absence of H, A, and B antigens.
  • No agglutination with anti-A, anti-B, or anti-H.

• Antibody Presence:

  • Presence of anti-H, anti-A, and anti-B in the serum.

• Saliva Composition:

  • No A, B, or H substances present in saliva.

• Compatibility:

  • Incompatible with any ABO blood groups.
  • Compatible with the Bombay phenotype only.

• Mode of Inheritance:

  • Follows a recessive mode of inheritance.
  • Identical phenotypes observed in children but not in parents.

ABO Subgroups

• Antigen Variation:

  • ABO subgroups differ in the amount of antigen present on the red blood cell membrane.
  • Subgroups have a reduced amount of antigen.

• Enzyme Efficiency:

  • Subgroups are the result of less effective enzymes.
  • Less efficient in converting H antigens to A or B antigens, resulting in fewer antigens on the RBC.

• Commonality:

  • Subgroups of A are more common than subgroups of B.

Blood Grouping

1. Test Unknown Cells with Known Antibodies:

   • Determine the blood group of unknown cells by testing them with known antibodies.

2. Test Unknown Serum/Plasma with Known Red Cells:

   • Determine the blood group of unknown serum/plasma by testing it with known red cells.

Blood Sample for Blood Grouping:

• Clearly labeled blood samples in sterile tubes (plain & EDTA).
• Perform the test on a fresh sample for the best results.
• If immediate testing is not possible, store the sample at 4oC and test within 48 hours.
• No signs of hemolysis should be present.
• If serum is not completely separated, centrifuge the tube at 3000 rpm for 3 min.
• Preferably use saline-washed red cells to make a 2-5% suspension.

Red Cell Suspensions for Blood Grouping:

• 2-5%: Test Tube Method
• 0.8-1%: Gel Technology
• 1%: Microplate

Test Tube Method of ABO Grouping:

Two Steps:

1. Cell Grouping (Forward Grouping):
   • Tests the patient's red cells with known Anti-A and Anti-B to determine the expressed antigen.
2. Serum Grouping (Reverse Grouping):
   • Tests the patient's serum with known A and B cells to determine the presence of antibodies.

1. Mix 1 volume of 2-5% red cell suspension with 2 volumes of Anti-A/Anti-B/Anti-AB.
2. Incubate at room temperature for 5 minutes.
3. Centrifuge at 1000 rpm for 1 minute.
4. Check for agglutination against a well-lighted background.

Reverse Grouping:

1. Mix 2 volumes of test serum/plasma with 1 volume of 5% suspension of reagent red cells.
2. Incubate at room temperature for 5 minutes.
3. Centrifuge at 1000 rpm for 1 minute.
4. Check for agglutination similarly as in cell grouping.

Tube Agglutination Grading:

• Microplate Method is ideal for testing a large number of blood samples.
• More sensitive to detect weaker antigen-antibody reactions.
• Results can be photographed for archival storage.
• Microplates can be incubated and centrifuged.
• Reusable after proper cleaning to remove foreign proteins.
• Adaptable for automation.

Rh (D) Antigen:

• Rh is a blood group system with many antigens, and one of them is D.
• Rh refers to the presence or absence of the D antigen on red blood cells.
• Individuals lacking the D antigen do not naturally produce anti-D.
• Antibody production to D requires exposure to the antigen.
• The D antigen is highly immunogenic; individuals exposed to it are likely to produce antibodies.
• All individuals are typed for D; if negative, they must receive Rh (D) negative blood.

Rh (D) Antigen Characteristics:

• Rh antigens are integral parts of the red cell membrane.
• Protein in nature with an active phospholipid component.
• Not present in soluble form; not excreted in body fluids.
• Unlike ABO antigens, Rh antigens are specific to red blood cells and are absent on platelets and leukocytes.

Rh (D) Antigen Frequency:

• Very potent antigen; around 50% may form antibodies upon exposure.
• In the Indian population, approximately 92-95% are Rh positive.
• Critical consideration for females of child-bearing age.
• Rh negative women are given anti-D injection after the birth of an Rh-positive baby to prevent immune response.

Rh Antibodies:

• All Rh antibodies are immune and developed after an immunizing event.
• React at 37°C and require an antiglobulin test to demonstrate the reaction.
• Generally, do not react at room temperature in saline.
• Mostly IgG in nature, capable of crossing the placenta.
• Generally, do not fix complement and cause extravascular hemolysis.
• Significant in Hemolytic Disease of the Newborn (HDN) and delayed Hemolytic Transfusion Reactions (HTR).

Rh Typing:

• Rh typing focuses on Rh (D).
• The result determines the Rh status (positive or negative).
• Some Rh typing sera are diluted in high-protein solutions and may require a negative control.
• Recommended to use two monoclonal anti-D sera (D1 and D2) from different manufacturers to confirm all Rh negatives.

Monoclonal Anti-D

1. IgM Anti-D Monoclonal Reagent:

   • Highly specific and saline-reacting equally at room temperature (RT) and 37°C.
   • Unreliable for detecting weak D.

2. Blend of IgM and IgG Monoclonal Antibodies Reagent:

   • Blended antibodies are routinely used and can detect weak D.

3. Monoclonal IgG Anti-D:

   • Used for Rh typing.

1. Preparation:

   • Prepare a 5% washed red cell suspension of the test sample.
   • Label three test tubes: Tubes 1 & 2 as "test" and Tube 3 as "control."
   • Place 1 drop of anti-D (D1) in Tube 1 and 1 drop of anti-D (D2) in Tube 2.
   • Place 1 drop of 22% bovine albumin (or control) in Tube 3.
   • Add 1 drop of 5% test cell suspension to each tube.

2. Mixing and Centrifugation:

   • Mix well and centrifuge at 1000 rpm for 1 minute.

3. Observation:

   • Resuspend the cell button and look for agglutination.
   • The control tube should show no agglutination.

4. RhD Negative Test on Blood Donor:

   • For all RhD-negative tests on blood donors, Du testing is recommended.

Note: Monoclonal antibodies are employed for Rh typing to enhance specificity and sensitivity, and the blending of IgM and IgG antibodies has become a common and reliable practice. The Du test is recommended to identify weak D phenotypes in RhD-negative individuals.

Method for Weak D Testing

1. Add 1 drop of 2-5% suspension of D-negative red cells to a test tube.
2. Add 2 drops of Anti-D (blend of IgG + IgM).
3. Incubate at 37°C for 30 minutes.
4. Wash three times with normal saline.
5. Make a dry red cell button and add polyspecific Anti-Human Globulin (AHG) reagent.
6. Look for agglutination.

Results:

• If there is agglutination, Du is Positive.
• If there is no agglutination, Du is Negative.

Significance of Weak D

Donors:

• Weak D testing on donors is required.
• Labeled as D positive but considered as recipient D negative.

Patients:

• Weak D testing on patients is not required.
• Standard practice is to transfuse with D-negative blood.

Additional Information:

• Donor Units: Weak D-positive donor units are labeled as Rh positive.
• Recipient Classification: Weak D-positive recipients are classified as Rh negative and safely transfused with Rh-negative blood.
• Antigenicity: Weak D is less antigenic than D, but transfusing weak D-positive red cells to a patient with anti-D can result in destruction. Hence, caution is exercised.
• Hemolytic Disease of the Newborn (HDN): Du-positive infants can be affected by HDN if the mother has anti-D antibodies.
• Rh Immunoprophylaxis: Rh immunoprophylaxis is recommended for Rh-negative mothers if the newborn is Du positive.